After treating cells with EPS, cells were harvested and lysed in RIPA buffer (Applygen Technology, Beijing, China) containing proteinase inhibitors and phosphatase inhibitors (ROCHE, Basel, Switzerland). BCA kit (Thermo Fisher Scientific, Rockford, IL, United States) was used to measure the protein concentration. Total of 70 μg amounts of protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes by wet blotting. The membranes were blocked in 10% non-fat dry milk for 1 h and then probed with antibodies against MCM2 (1:1,000, ABclonal, Boston, MA, United States), Caspases-3 (1:1,000, CST, Boston, MA, United States), PARP (1:1,000, CST, Boston, MA, United States), cleaved-PARP (1:1,000, CST, Boston, MA, United States) and RPS18 (1:1,000, ABclonal, Boston, MA, United States) separately at 4°C overnight. Followed by incubation with peroxidase-linked secondary antibodies (1:10,000, CST, Boston, MA, United States) for 1 h at room temperature, the enhanced chemiluminescent (ECL) reagent (Thermo Fisher Scientific, Rockford, IL, United States) was used to visualize the immunoreactive proteins.
Proteinase inhibitors and phosphatase inhibitors
Manufactured by Roche
Sourced in Switzerland
Proteinase inhibitors and phosphatase inhibitors are laboratory reagents used to prevent the degradation of proteins and the dephosphorylation of phosphorylated molecules, respectively, during sample preparation and analysis. They are commonly used in biochemical, cell biology, and proteomics research to maintain the integrity of proteins and preserve post-translational modifications.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rps18, by ABclonal (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Applygen (1 mentions)
Ab10275, by Abcam (1 mentions)
Ap132p, by Merck Group (1 mentions)
Ap124p, by Merck Group (1 mentions)
A1978, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
P65 af5006, by Affinity Biosciences (1 mentions)
P p65 af2006, by Affinity Biosciences (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bradford assay, by Beyotime (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Westernbright ecl reagent, by Advansta (1 mentions)
7 protocols using proteinase inhibitors and phosphatase inhibitors
1
Protein Expression Analysis of EPS-Treated Cells
2
Spinal Cord and DRG Protein Analysis in Anesthetized Rats
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Rats were deeply anesthetized with 2% pentobarbital (60 mg/kg, i.p.). The dorsal part of the L4–L5 spinal segments and L4–L5 DRG were removed immediately and homogenized in lysis buffer containing proteinase inhibitors and phosphatase inhibitors (Roche, Switzerland). The protein concentration was tested using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Proteins were separated by polyacrylamide gel electrophoresis. The antibodies used were as follows: rabbit-anti-MOR antibody (1:1000; ab10275; Abcam), mouse anti-β-actin antibody (1:5000; A1978; Sigma-Aldrich), peroxidase-conjugated goat anti-rabbit IgG (1:5000; AP132P; Millipore), and peroxidase-conjugated goat anti-mouse IgG (1:5000; AP124P; Millipore). The protein bands were visualized with an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA) and detected by the ChemiDoc Imaging System (Bio-Rad, Berkeley).
3
Western Blot Analysis of MTA1, p65, and p-p65
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Western blot assay was performed as previously described [16 (link)]. In brief, the treated A498 cells were lysed with RIPA lysis buffer containing proteinase inhibitors and phosphatase inhibitors (Roche). Then, the obtained cell lysates were used for western blot analysis. The MTA1 (BA2749) was from BOSTER, and β-actin (10230-1-AP) antibodie was purchased from Proteintech, and p65 (AF5006) and p-p65 (AF2006) antibodies were obtained from Affinity Biosciences.
4
Quantification of Endoplasmic Reticulum Stress Markers
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qRT-PCR was done as previously described (23 (link)). Total RNAs were isolated and purified from orbital adipose/connective sample using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA was synthesized utilizing the PrimeScript RT Reagent Kit (Takara, Otsu, Shiga, Japan) following the manufacturer’s instructions. Gene expression level was quantified by qRT-PCR using SYBR Premix Taq (Takara, Otsu, Shiga, Japan) and normalized to β-actin. All primer sequence information is listed in Table 2
The tissue was lysed by RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was evaluated by BCA protein assay kit (Vayme Biotechnology, Nanjing, China). The proteins were separated on 4-16% gradient SDS-PAGE, and then transferred to PVDF membrane (Millipore, Bedford, MA, USA). The detection of chemiluminescence was performed as previously described (24 (link)).Western blot results were quantified using ImageJ (http://rsb.info.nih.gov/ij/index.html ). Primary antibodies involved in IRE1α (CST, cat.No: 3294, 1:1000), PERK (CST, cat.No: 5683, 1:1000), ATF6 (CST, cat.No: 65880, 1:1000), and β-Actin (CST, cat.No: 4967, 1:1000) were used for western blot.
The tissue was lysed by RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was evaluated by BCA protein assay kit (Vayme Biotechnology, Nanjing, China). The proteins were separated on 4-16% gradient SDS-PAGE, and then transferred to PVDF membrane (Millipore, Bedford, MA, USA). The detection of chemiluminescence was performed as previously described (24 (link)).Western blot results were quantified using ImageJ (
5
Western Blot Analysis of Protein Expression
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We used protein lysis buffer (Beyotime Institute of Biotechnology) together with the appropriate concentrations of proteinase inhibitors and phosphatase inhibitors (Roche Applied Science, Indianapolis, IN, USA) to extract cell proteins. Then, the Bradford assay (Beyotime Institute of Biotechnology) was used to detect the total protein concentration. The protein samples were fractionated by SDS-PAGE (12% polyacrylamide gels) followed by transfer onto nitrocellulose membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). After incubation in blocking buffer (20 mM Tris, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) containing 5% nonfat dry milk powder, the membranes were incubated with the indicated antibodies overnight at 4°C, followed by reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Enhanced chemiluminescent reagents were used to visualize the protein bands by chemiluminescence detection kit. Beta-actin was used as an internal control.
6
Western Blot Protein Analysis Protocol
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Proteins of cells or tissue samples were obtained by RIPA lysis buffer (Beyotime biotechnology, China), which was added in proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The Bradford method was applied to quantify the protein samples. After then, 30 μg of protein was loaded to SDS-PAGE before transferring to nitrocellulose membranes (Bio-Rad Biotechnology, America). The membranes were blocked by TBST buffer containing 2.5% skim milk for 30 min. Then, the membranes were incubated with primary antibody at 4 °C overnight. Next day, the peroxidase-conjugated secondary antibody was added to incubate the membrane, and blot images were acquired by an enhanced chemiluminescence kit.
7
Western Blot Analysis of MAPK Signaling
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Cell lysate was extracted from RAW264.7 cells using RIPA lysis buffer (Beyotime, China) containing proteinase inhibitors and phosphatase inhibitors (Roche, Germany). The lysate was separated in 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Primary antibodies for Western blotting analysis were ordered from Cell Signaling Technology (USA), including monoclonal mouse anti-phospho-extracellular signal-regulated kinase 1/2 (Erk1/2) (#9106), polyclonal rabbit anti-Erk1/2 (#9102), monoclonal rabbit anti-phospho-p38 (#4511), monoclonal rabbit anti-p38 (#8690), monoclonal rabbit anti-phospho-c-Jun N-terminal kinase (JNK) (#4668), polyclonal rabbit anti-JNK (#9252), monoclonal rabbit anti-phospho-mitogen-activated protein kinase kinase 1/2 (MEK1/2) (#2338), monoclonal mouse anti-MEK1/2 (#4694), and monoclonal rabbit anti-β-tubulin antibody (#2128). After primary antibody incubation, horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (Promega, USA) was applied. WesternBright ECL reagent (Advansta, USA) was used to visualize the signal, which was then scanned using an UVP BioSpectrum imaging system (BioSpectrum 410, USA). Densitometry analysis was performed using Image J.
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